Rapid and Multiplexed MicroRNA Diagnostic Assay Using Quantum Dot-Based Förster Resonance Energy Transfer

Abstract

The detection of next generation microRNA (miRNA) biomarkers has become a highly important aspect for clinical diagnostics. We use multiplexed Förster resonance energy transfer (FRET) between a luminescent Tb complex and three different semiconductor quantum dots (QDs) to sensitively detect three different miRNAs from a single 150 μL sample with ca. 1 nM (subpicomol) detection limits. The rapid and amplification-free mix-and-measure assay format is based on careful design of miRNA base pairing and stacking to selectively detect different miRNAs with very strong sequence homologies. Clinical applicability is demonstrated by sensitive multiplexed quantification of three miRNAs at low (2 to 10 nM) and varying concentrations in samples that contained up to 10% serum.

Emerging Biosensing Approaches for microRNA Analysis

“…Here, three miRNAs were detected in diluted serum without the need for any washing steps, and a LOD of 1 nM was reported. 55 …”

“…(B) Quantum dot FRET pairs were also used to profile three miRNAs from diluted serum. Reproduced from Qiu, X.; Hildebrandt, N. ACS Nano 2015, 9, 8449–8457 (ref 55). Copyright 2015 American Chemical Society.…”

Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications

“…Based on this type of design criteria, a recently prototyped diagnostic sensor for the multiplexed detection of miRNA used three different SAv-coated QDs (Qdots, Life Technologies) in combination with biotinylated DNA probes. 631 As shown in Figure 73, the probes exploited base pairing and stacking effects, which led to the formation of three different Tb/QD FRET pairs only in the presence of specific miRNA targets. The biotinylated-DNA probes attached to the QDs were designed to be too short (7 or 8 bases) for efficient hybridization with the complementary sequence of the Tb-reporter oligonucleotides (Figure 73A left).…”

miRNA assays in the clinical laboratory: workflow, detection technologies and automation aspects

“…Recently, a plethora of novel assay concepts for measurement of miRNAs has been published, including formats based on multiplexed Förster or flourescence resonance energy transfer (FRET) and quantum dot assay designs [116][117][118], and ligation-mediated hybridization to microarrays [119], respectively. It remains to be elucidated if any of these methods can take the next step to move into the clinical routine.…”